Two forms of human cytoplasmic arginyl-tRNA synthetase produced from two translation initiations by a single mRNA

Human cytoplasmic arginyl-tRNA synthetase (ArgRS) is a component of a macromolecular complex consisting of at least nine tRNA synthetases and three auxiliary proteins. In mammalian cells, ArgRS is present as a free protein as well as a component of the complex. Via an alignment of ArgRSs from different vertebrates, the genes encoding full-length human cytoplasmic ArgRS and an N-terminal 72-amino acid deletion mutant (hcArgRS and ΔNhcArgRS, respectively) were subcloned and expressed in Escherichia coli. The two ArgRS products were expressed as a soluble protein in E. coli. The level of production of ΔNhcArgRS in E. coli and its specific activity were higher than those for hcArgRS. By Western blot analysis, using an antibody against the purified ΔNhcArgRS, the two forms of ArgRS were detected in three human cell types. The 5′-end cDNA sequence, as confirmed by 5′RACE (5′-rapid amplification of cDNA ends), contained three start codons. Through mutation of the three codons, the two human cytoplasmic ArgRSs were found to be produced in different amounts, indicating that they resulted from two different translation initiation events. Here we show evidence that two forms of human cytoplasmic ArgRS were produced from two translational initiations by a single mRNA.